Development of the mammalian preimplantation embryo is influenced by autocrine/paracrine factors and availability of nutrients. Deficiencies of these during in vitro culture reduce the success of assisted reproductive technologies. The mTORC1 pathway integrates external and internal signals, including those by amino acids (AAs), to promote normal preimplantation development. For this reason, AAs are often included in embryo culture media. In this study, we examined how withdrawal and addition of AAs to culture media modulate mTORC1 pathway activity compared to its activity in mouse embryos developed in vivo. Phosphorylation of signalling components downstream of mTORC1, namely p70S6K, RPS6 and 4E-BP1, and that of Akt, which lies upstream of mTORC1, changed significantly across stages of embryos developed in vivo. For freshly isolated blastocysts placed in vitro, the absence of AAs in culture medium, even for a few hours, decreased mTORC1 signalling, which could only be partially restored by their addition. Long-term culture of early embryos to blastocysts in the absence of AAs decreased mTORC1 signalling to a greater extent and again this could only be partially restored by their inclusion. This failure to fully restore is probably due to decreased PI3K/Akt/mTORC2 signalling in culture, as indicated by decreased P-Akt^S473. mTORC2 lies upstream of mTORC1 and is insensitive to AAs, and its reduced activity probably results from loss of maternal/autocrine factors. These data highlight reduced mTORC1/2 signalling activity correlating with compromised development in vitro and show that addition of AAs can only partially offset these effects.

中文翻译：

---

小鼠植入前胚胎的无氨基酸培养可下调mTORC1 / 2信号传导，而氨基酸重新添加仅可部分恢复mTORC1 / 2信号传导

哺乳动物植入前胚胎的发育受自分泌/旁分泌因子和养分利用率的影响。这些在体外培养过程中的缺陷降低了辅助生殖技术的成功率。mTORC1途径整合了外部和内部信号，包括氨基酸（AAs）的信号，以促进正常的植入前发育。因此，AA通常包含在胚胎培养基中。在这项研究中，我们检查了与在体内发育的小鼠胚胎中的mTORC1途径活性相比，向培养基中撤除和添加AA如何调节mTORC1途径活性。mTORC1下游信号传导成分的磷酸化，即p70S6K，RPS6和4E-BP1，以及mTORC1上游的Akt的磷酸化，在体内胚胎发育的各个阶段都发生了显着变化。对于体外放置的新鲜分离的胚泡，即使在几个小时内在培养基中都不存在AA，也会降低mTORC1信号传导，只能通过添加它们才能部分恢复。在缺乏AA的情况下，将早期胚胎长期培养为胚泡会在更大程度上降低mTORC1信号传导，并且再次通过包涵可以部分恢复。无法完全恢复的失败可能是由于培养物中PI3K / Akt / mTORC2信号转导减少所致，如P-Akt减少 在缺乏AA的情况下，将早期胚胎长期培养为胚泡会在更大程度上降低mTORC1信号传导，并且再次通过包涵可以部分恢复。无法完全恢复的失败可能是由于培养物中PI3K / Akt / mTORC2信号转导减少所致，如P-Akt减少 在缺乏AA的情况下，将早期胚胎长期培养为胚泡会在更大程度上降低mTORC1信号传导，并且再次通过包涵可以部分恢复。无法完全恢复的失败可能是由于培养物中PI3K / Akt / mTORC2信号转导减少所致，如P-Akt减少^S473。mTORC2位于mTORC1的上游，对AA不敏感，其活性降低可能是由于母体/自分泌因子的丧失。这些数据强调了与体外发育受损相关的mTORC1 / 2信号活性降低，并表明添加AA只能部分抵消这些影响。